GLP-1 analogues

ABSTRACT

The present disclosure pertains to novel Glucagon like Peptide-1 (GLP-1) (7-37) analogs having an amino acid sequence with Leu or Ile at the C-terminal. The new analogs are potent GLP-1 agonists with reduced adverse effect and improved duration of action. The present disclosure further relates to acylated derivatives of the new analogs which have further improved potency and duration of action and are suitable for oral administration. The analogs of present disclosure may be useful in treatment of diabetes and obesity.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/134,982, filed Dec. 28, 2020, which is a continuation of U.S.application Ser. No. 16/376,190, filed Apr. 5, 2019, which claims thebenefit of three Indian provisional applications having application nos.IN 201821013109 (filed on Apr. 5, 2018); IN 201821040468 (filed on Oct.26, 2018) and IN 201821040474 (filed on Oct. 26, 2018), all of which arehereby incorporated by reference.

SEQUENCE LISTING STATEMENT

A computer readable form of the Sequence Listing is filed with thisapplication by electronic submission and is incorporated into thisapplication by reference in its entirety. The sequence listing submittedherewith is contained in the file created Jul. 27, 2022, entitled“19-678-USCON4_SequenceListing.xml” and is 299 kilobytes in size.

FIELD OF THE DISCLOSURE

The present disclosure relates to novel Glucagon like Peptide-1 (GLP-1)(7-38) analogs having an amino acid sequence with Leu or Ile at theC-terminal. The new analogs are potent GLP-1 agonists with reducedadverse effect and improved duration of action. The present disclosurefurther relates to acylated derivatives of the new analogs, which havefurther improved potency and duration of action and are suitable fororal administration. The analogs disclosed herein are acylated withprotracting moieties, which increase the duration of activity of thecompounds. The analogs disclosed herein may be useful in treatment ofdiabetes and obesity.

BACKGROUND OF THE DISCLOSURE

Glucagon-like peptide-1 (GLP-1) is a hormone that is mainly produced inenteroendocrine cells of the gut and is secreted into the blood streamwhen food containing fat, protein hydrolysate and/or glucose enters theduodenum. GLP-1 is derived from the cell-specific post-translationalprocessing of the preproglucagon gene. Initially, the peptideGLP-1(1-37) was identified from this processing, but it was the twoN-terminally truncated products, GLP-1(7-37) (SEQ ID NO: 1) andGLP-1(7-36) amide, that were found to recognize the pancreatic receptorand which were determined to be the active species in vivo. GLP-1 hasbeen found to stimulate insulin secretion, thereby causing glucoseuptake by cells and decreased serum glucose levels. GLP-1 agonists areavailable for the treatment for Type 2 Diabetes Mellitus (T2DM) asfavored drugs as they are devoid of hypoglycemia and with a positivebenefit of weight loss. The endogenous substance, GLP-1(7-37) andGLP-1(7-36)amide, are cleaved by peptidases and thus have a very shorthalf-life. Efforts were made to improve the performance by developingGLP-1 analogues with improved half-life. The first drug approved in 2005was Exenatide with twice a day dosing at dose level 10 mcg and was foundto show a significant improvement in HbA1c, a marker of glucose control.Further, Novo Nordisk developed Liraglutide (U.S. Pat. No. 6,268,343)(SEQ ID NO: 2) with once a day dosing of 1.8 mg, s.c./day and approvedin 2010. Further research and development produced once a week productslike, Albiglutide developed by GSK and Dulaglutide developed by EliLilly. Recently, Semaglutide (International Publication No. WO2006/097537 A2), a GLP-1 analogue was approved by USFDA. Semaglutide(SEQ ID NO: 3) is marketed under the brand name Ozempic®. It isadministered as a once-weekly subcutaneous injection.

Many attempts to make GLP-1 analogs having improved potency and durationof action are reported in literature. U.S. Pat. No. 7,291,594 B2 (the US'594 patent) discloses GLP-1 (7-35) derivatives having added severalresidues of arginine and/or lysine to the C-terminus thereof to providehigh bioavailability via mucous membranes. The US '594 patent furtherdiscloses that these derivative can be conferred with resistance todipeptidyl peptidase IV (DPP-IV) by substituting amino acid 8 in itsGLP-1 amino acid sequence with Ser, or with resistance to trypsin bysubstituting amino acids 26 and 34 with Gln and Asn, respectively. U.S.Pat. No. 7,893,017 B2 (the US '017 patent) discloses acylated GLP-1analog wherein the GLP-1 analog is stabilized against DPP-IV bymodification of at least one amino acid residue in positions 7 and 8relative to the sequence GLP-1 (7-37) and wherein said acylation is adiacid attached directly to the C-terminal amino acid residue of saidGLP-1 analog.

U.S. Pat. No. 8,951,959 B2 (the US '959 patent) discloses a DPP-IVresistant GLP-1 (7-37) analogue having a non-proteogenic amino acidresidue containing trifluromethyl group in position 8 relative to thesequence GLP-1, and is acylated with a moiety comprising two acidicgroups to the lysine residue in position 26.

U.S. Pat. No. 7,084,243 B2 (the US '243 patent) discloses GLP-1 (7-37)analogues having Val or Gly at position 8 relative to the sequence GLP-1(7-37) as DPP-IV resistant peptides.

International Publication No. WO 2017/149070 A1 (the WO '070) disclosesGLP-1 analogues having a Trp at a position corresponding to position 8of GLP-1 (7-37) and these Trp8 compounds were shown to be very stableagainst degradation by DPP-IV.

International Publication No. WO 2004/103390A2 (the WO '390) disclosesthat the modification at the P′₁ position (corresponding to 9 positionin case of GLP-1 (7-37)) can produce GLP-1 analogues with greatlyreduced susceptibility to enzyme-mediated (such as DPP-IV) cleavagerelative to the native substrate, yet retain the biological activity ofthe native substrate. The WO '390 further discloses GLP-1 (7-37)analogues having an amino acid with tetrasubstituted CP carbon (such astert-leucine) at position 9 provides GLP-analogues with resistant todegradation by DPP-IV.

International Publication No. WO 2015/086686 A2 (the WO '686publication) discloses that incorporation of alpha-methyl-functionalizedamino acids directly into the main chain of GLP-1 analogues has beendetermined to produce protease-resistant (includes DPP-IV resistant)peptides.

Various other DPP-IV resistant GLP-1 agonists are disclosed in patentpublications such as International Publication Nos. WO 2007/030519 A2,WO 2004/078777 A2, WO 2007/039140 A1, WO 2014/209886 A1, WO 2012/016419A1, WO 2017/211922 A2, WO 2016/198544 A1 and WO 2013/051938 A2.

Various patent applications disclose C-terminally extended GLP-1analogues with increased stability and longer duration of action. Forexample, U.S. Pat. Nos. 7,482,321 B2, 9,498,534 B2 and 7,897,566 B2.

Various patent applications disclose acylated GLP-1 analogs wherein theGLP-1 analogues are attached to lipophilic substituent optionally via alinker to provide longer duration of action.

U.S. Pat. No. 8,603,972 B2 (the US '972) discloses monoacylatedderivatives of GLP-1 analogues wherein Lys residue at position 37 or 38of GLP-1 analogue is acylated. U.S. Pat. Nos. 8,648,041 B2, 9,758,560B2, 9,006,178 B2, 9,266,940 B2, 9,708,383 B2 and United States PatentApplication Publication Nos. US 2015/0152157 A1, US 2015/0133374 A1disclose di-acylated derivatives of GLP-1 analogues.

United States Patent Application Publication No. US 2016/0200791 A1discloses triacylated derivatives of GLP-1 analogues.

International Publication Nos. WO 2016/083499 A1, WO 2016/097108 A1 andWO 2014/202727 A1 disclose acylated GLP-1 analogues wherein the Lysresidue of GLP-1 analogues is attached to two protracting moieties via abranched linker.

International Publication Nos. WO 2009/030771 A1 and WO 2018/083335 A1disclose various acylating agents (side chain) which can be attached toLys residue of GLP-1 analogues to provide longer duration of action.

International Publication No. WO 2013/186240 A2 discloses exendin-4peptide analogues having Gly, Ser or functionalized Ser, e.g., Ser(OCH₃), D-Ser or functionalized D-Ser, e.g., D-Ser(OCH₃), Aib, Ala, orD-Ala at position 2 of exendin-4 amino acid sequence.

Various other GLP-1 analogues are disclosed in patent applications suchas International Publication Nos. WO 2005/027978 A2, WO 1998/008871 A1,WO 1999/043705 A1, WO 1999/043706 A1, WO 1999/043707 A1, WO 1999/043708A1, WO 2000/034331 A2, WO 2009/030771 A1, WO 2011/080103 A1, WO2012/140117 A1, WO 2012/062803 A1, WO 2012/062804 A1, WO 2013/037690 A1,WO 2014/202727 A1, WO 2015/000942 A1, WO 2015/022400 A1, WO 2016/083499A1, WO 2016/097108 A1 and WO 2017/149070 A1.

Still, there is need to develop GLP-1 analogs which have optimum desiredproperties in terms of stability and duration of action.

SUMMARY OF THE DISCLOSURE

One aspect the present disclosure provides a polypeptide comprising theamino acid sequence:

-   -   H-X2-X3-X4-G-T-F-T-S-D-V-S-S-Y-L-X16-G-Q-A-A-X21-E-F-X24-A-W-L-V-R-G-R-G-X33-X34    -   wherein X2 is Ser, Ser(OMe), D-Ser, D-Ser(OMe), Ala, or Aib;    -   X3 is absent or Gln;    -   X4 is Glu;    -   X16 is Glu;    -   X24 is Ile;    -   X33 is Leu, D-Leu, D-Ile, or Ile;    -   X34 is absent and    -   X21 is Lys wherein the side chain amino (ε amino) group of Lys        is acylated with a moiety:        {-Q-T-U—W—Y—Z    -   wherein Q and T are absent;    -   U is absent or —C(O)—CH₂—O—(CH₂)₂—O—(CH₂)₂—NH—} wherein } is        point of attachment with group W;    -   W is absent or selected from a group consisting of        —C(O)—CH₂—O—(CH₂)₂—O—(CH₂)₂—NH—], —C(O)—NH—(CH₂)₃₋₄—NH—],        —C(O)—C(CH₃)₂—NH—] and

-   -    wherein ] is point of attachment with group Y;    -   Y is —C(O)—(CH₂)₂—CH(COOH)NH—, wherein — is point of attachment        with the group Z;    -   Z is —C(O)—(CH₂)_(n)—COOH or —C(O)—(CH₂)_(n)—CH₃ wherein n is an        integer from 14 to 20.

The polypeptides of the present disclosure are potent GLP-1 agonistswith fewer adverse effects. Further, the polypeptides of the presentdisclosure are stable and have long duration of action and are suitablefor oral administration.

DESCRIPTION OF FIGURES

FIG. 1A illustrates the preparation of Moiety A-OSu (Intermediate 3).FIG. 1B illustrates the preparation of Moiety A-OSu (Intermediate 3).

FIG. 2 illustrates the preparation of Moiety C-OSu.

FIG. 3 illustrates the preparation of Moiety D-OSu.

FIG. 4 illustrates the preparation of Moiety E-OSu.

FIG. 5 illustrates the preparation of Moiety F-OSu.

FIGS. 6A and 6B illustrate the results of an oral glucose tolerance test(OGTT) for Compound 1 in rats; single injection; 1 mg/kg glucose AUC0-120 min (FIG. 6A=After 22 hrs, FIG. 6B=After 46 hrs).

FIG. 7 illustrates the reduction in blood glucose levels in db/db type 2diabetic mice after chronic treatment with Compound 1.

FIG. 8 illustrates the reduction in food intake in db/db mice followingtreatment with Compound 1.

FIG. 9 illustrates the efficacy of Compound 1 in db/db mice in reductionof body weight.

FIG. 10 illustrates the reduction in Hb1Ac in db/db mice followingtreatment with Compound 1.

ABBREVIATIONS

-   -   Aib: 2-Aminoisobutyric acid    -   ADO: 8-Amino-3,6-dioxo-octanoic acid    -   OGTT: Oral glucose tolerance test    -   DIPEA: N,N′-Di-isopropylethylamine    -   HOBt: 1-Hydroxybenztriazole    -   DIPC: N,N′-Di-isopropylcarbodbmide    -   HOSu: N-Hydroxysuccinimide    -   IBCF: Isobutyl chloroformate    -   NMM: N-Methylmorpholine    -   THF: Tetrahydrofuran    -   DCM: Dichloromethane    -   DMAP: 4-Dimethylaminopyridine    -   DCC: Dicyclohexyl carbodiimide    -   DMAc: Dimethylacetamide

DESCRIPTION OF THE DISCLOSURE

The present disclosure provides a stable long acting GLP-1 analog, whichdoes not require frequent subcutaneous dosing and is also suitable fororal administration. It was surprisingly found that the addition of anextra Leu at the C terminal of the sequence produced peptides withsignificantly improved potency and duration of action when compared tothe parent peptide. The peptides with an extra Ile also showed similareffect of improved potency and duration of action when compared to theparent peptide. Additionally, the disclosure herein demonstratesmoieties, which can be appended to peptides which are analogs ofGLP-1(7-37) via acylation reaction to produce compounds withsignificantly improved potency and longer duration of action. Theprotracting moieties of the disclosed compounds have more stable bonds,which are less susceptible to cleavage by biological enzymes. Thus, thecompounds disclosed herein are more stable and require less frequentadministration adding to patient compliance. Accordingly, in someembodiments, the disclosure provides a polypeptide comprising the aminoacid sequence:

(SEQ ID NO: 4) H-X2-X3-X4-G-T-F-T-S-D-V-S-S-Y-L-X16-G-Q-A-A-X21-E-F-X24-A-W-L-V-R-G-R-G-X33-X34

-   -   wherein X2 is Ser, Ser(OMe), D-Ser, D-Ser(OMe), Ala, or Aib;    -   X3 is absent or Gln;    -   X4 is Glu;    -   X16 is Glu;    -   X24 is Ile;    -   X33 is Leu, D-Leu, D-Ile, or Ile;    -   X34 is absent and    -   X21 is Lys wherein the side chain amino (ε amino) group of Lys        is acylated.

In some embodiments, X21 can be acylated with the protracting moietiesreported in U.S. Pat. Nos. 6,268,343, 8,951,959 B2, 8,603,972 B2,8,648,041 B2, 9,758,560 B2, 9,006,178 B2, 9,266,940 B2, 9,708,383 B2 andUnited States Patent Application Publication Nos. US 2015/0152157 A1 andUS 2015/0133374 A1; International Publication Nos. WO 2009/030771 A1, WO2006/097537 A2 and WO 2018/083335 A1.

In some embodiments, the X21 Lys is acylated at its side chain amino (εamino) group with a moiety comprising a fatty acid group. The fatty acidgroup may be attached to X21 Lys via a linker. Accordingly, in someembodiments, the present disclosure provides a polypeptide comprisingthe amino acid sequence:

-   -   H-X2-X3-X4-G-T-F-T-S-D-V-S-S-Y-L-X16-G-Q-A-A-X21-E-F-X24-A-W-L-V-R-G-R-G-X33-X34    -   wherein X2 is Ser, Ser(OMe), D-Ser, D-Ser(OMe), Ala, or Aib;    -   X3 is absent or Gln;    -   X4 is Glu;    -   X16 is Glu;    -   X24 is Ile;    -   X33 is Leu, D-Leu, D-Ile, or Ile;    -   X34 is absent and    -   X21 is Lys wherein the side chain amino (ε amino) group of Lys        is acylated with a moiety:        {-Q-T-U—W—Y—Z    -   wherein Q and T are absent;    -   U is absent or —C(O)—CH₂—O—(CH₂)₂—O—(CH₂)₂—NH—} wherein } is        point of attachment with group W;    -   W is absent or selected from a group consisting of        —C(O)—CH₂—O—(CH₂)₂—O—(CH₂)₂—NH—], —C(O)—NH—(CH₂)₃₋₄—NH—],        —C(O)—C(CH₃)₂—NH—] and

-   -    wherein ] is point of attachment with group Y;    -   Y is —C(O)—(CH₂)₂—CH(COOH)NH—, wherein — is point of attachment        with the group Z;    -   Z is —C(O)—(CH₂)_(n)—COOH or —C(O)—(CH₂)_(n)—CH₃ wherein n is an        integer from 14 to 20.

In some embodiments, the amino acid at X2 is selected from Ser,Ser(OMe), D-Ser, D-Ser(OMe), Ala or Aib.

In some embodiments X2 is Aib.

In some embodiments, X3 is absent.

In some embodiments, X33 is Leu.

In some embodiments, X33 is Ile.

In some embodiments, X21 is Lys wherein the side chain amino (ε amino)group of Lys is acylated with a moiety:{-Q-T-U—W—Y—Z

-   -   wherein W is selected from a group consisting of        —C(O)—NH—(CH₂)₃₋₄—NH—], —C(O)—C(CH₃)₂—NH—] and

In some embodiments, X21 is Lys wherein the side chain amino (ε amino)group of Lys is acylated with a moiety:{-Q-T-U—W—Y—Z,

-   -   wherein U and W both are absent and Z is —C(O)—(CH₂)_(n)—CH₃        wherein n is an integer 14.

In some embodiments, X21 is Lys wherein the side chain amino (ε amino)group of Lys is acylated with a moiety:{-Q-T-U—W—Y—Z,

-   -   wherein W is —C(O)—CH₂—O—(CH₂)₂—O—(CH₂)₂—NH—].

In some embodiments, X21 is Lys wherein the side chain amino (ε amino)group of Lys is acylated with a moiety:{-Q-T-U—W—Y—Z,

-   -   wherein W is —C(O)—C(CH₃)₂—NH—].

In some embodiments, X21 is Lys wherein the side chain amino (ε amino)group of Lys is acylated with a moiety:{-Q-T-U—W—Y—Z,

-   -   wherein W is —C(O)—NH—(CH₂)₄—NH—].

In some embodiments, X21 is Lys wherein the side chain amino (ε amino)group of Lys is acylated with a moiety:{-Q-T-U—W—Y—Z,

-   -   wherein W is —C(O)—NH—(CH₂)₃—NH—].

In some embodiments, X21 is Lys wherein the side chain amino (ε amino)group of Lys is acylated with a moiety:{-Q-T-U—W—Y—Z,

wherein W is

In some embodiments, X21 is Lys wherein the side chain amino (ε amino)group of Lys is acylated with a moiety:{-Q-T-U—W—Y—Z,

-   -   wherein Z is —C(O)—(CH₂)_(n)—COOH, wherein n is integer 16.

In some embodiments, X21 is Lys wherein the side chain amino (ε amino)group of Lys is acylated with a moiety:{-Q-T-U—W—Y—Z,

-   -   wherein Z is —C(O)—(CH₂)_(n)—CH₃ wherein n is an integer 14.

In some embodiments, X2 is Ala or Aib;

-   -   X3 is absent;    -   X33 is Leu;    -   U is absent;    -   W is absent;    -   Y is —C(O)—(CH₂)₂—CH(COOH)NH—, wherein — is point of attachment        with the group Z;    -   Z is —C(O)—(CH₂)_(n)—CH₃ wherein n is integer 14.

In some embodiments, the present disclosure provides a polypeptidecomprising the amino acid sequence:

-   -   H-X2-X3-X4-G-T-F-T-S-D-V-S-S-Y-L-X16-G-Q-A-A-X21-E-F-X24-A-W-L-V-R-G-R-G-X33-X34    -   wherein X2 is Aib;    -   X3 is absent;    -   X4 is Glu;    -   X16 is Glu;    -   X24 is Ile;    -   X33 is Leu;    -   X34 is absent and    -   X21 is Lys wherein the side chain amino (ε amino) group of Lys        is acylated with a moiety:        {-Q-T-U—W—Y—Z    -   wherein Q and T are absent;    -   U is —C(O)—CH₂—O—(CH₂)₂—O—(CH₂)₂—NH—} wherein } is point of        attachment with group W;    -   W is —C(O)—CH₂—O—(CH₂)₂—O—(CH₂)₂—NH—], wherein ] is point of        attachment of group Y;    -   Y is —C(O)—(CH₂)₂—CH(COOH)NH—, wherein — is point of attachment        with the group Z;    -   Z is —C(O)—(CH₂)_(n)—COOH wherein n is integer 16.

In some embodiments, the present disclosure provides a polypeptidecomprising the amino acid sequence:

-   -   H-X2-X3-X4-G-T-F-T-S-D-V-S-S-Y-L-X16-G-Q-A-A-X21-E-F-X24-A-W-L-V-R-G-R-G-X33-X34    -   wherein X2 is Aib;    -   X3 is absent;    -   X4 is Glu;    -   X16 is Glu;    -   X24 is Ile;    -   X33 is Leu;    -   X34 is absent and    -   X21 is Lys wherein the side chain amino (ε amino) group of Lys        is acylated with a moiety:        {-Q-T-U—W—Y—Z    -   wherein Q and T are absent;    -   U is —C(O)—CH₂—O—(CH₂)₂—O—(CH₂)₂—NH-} wherein } is point of        attachment with group W;    -   W is —C(O)—C(CH₃)₂—NH—] wherein ] is point of attachment with        group Y;    -   Y is —C(O)—(CH₂)₂—CH(COOH)NH—, wherein — is point of attachment        with the group Z;    -   Z is —C(O)—(CH₂)_(n)—COOH wherein n is integer 16.

In some embodiments, the present disclosure provides a polypeptidecomprising the amino acid sequence:

-   -   H-X2-X3-X4-G-T-F-T-S-D-V-S-S-Y-L-X16-G-Q-A-A-X21-E-F-X24-A-W-L-V-R-G-R-G-X33-X34    -   wherein X2 is Aib;    -   X3 is absent;    -   X4 is Glu;    -   X16 is Glu;    -   X24 is Ile;    -   X33 is Leu;    -   X34 is absent and    -   X21 is Lys wherein the side chain amino (ε amino) group of Lys        is acylated with a moiety:        {-Q-T-U—W—Y—Z    -   wherein Q and T are absent;    -   U is —C(O)—CH₂—O—(CH₂)₂—O—(CH₂)₂—NH—} wherein } is point of        attachment with group W;    -   W is —C(O)—NH—(CH₂)₃₋₄—NH—] wherein ] is point of attachment        with group Y;    -   Y is —C(O)—(CH₂)₂—CH(COOH)NH—, wherein — is point of attachment        with the group Z;    -   Z is —C(O)—(CH₂)_(n)—COOH wherein n is integer 16.

In some embodiments, X21 is Lys wherein the side chain amino (ε amino)group of Lys is acylated with a moiety:{-Q-T-U—W—Y—Z,

-   -   wherein W is —C(O)—NH—(CH₂)₄—NH—].

In some embodiments, X21 is Lys wherein the side chain amino (ε amino)group of Lys is acylated with a moiety:{-Q-T-U—W—Y—Z,

-   -   wherein W is —C(O)—NH—(CH₂)₃—NH—].

In some embodiments, the disclosure provides a polypeptide comprisingthe amino acid sequence:

-   -   H-X2-X3-X4-G-T-F-T-S-D-V-S-S-Y-L-X16-G-Q-A-A-X21-E-F-X24-A-W-L-V-R-G-R-G-X33-X34    -   wherein X2 is Aib;    -   X3 is absent;    -   X4 is Glu;    -   X16 is Glu;    -   X24 is Ile;    -   X33 is Leu;    -   X34 is absent and    -   X21 is Lys wherein the side chain amino (ε amino) group of Lys        is acylated with a moiety:        {-Q-T-U—W—Y—Z    -   wherein Q and T are absent;    -   U is —C(O)—CH₂—O—(CH₂)₂—O—(CH₂)₂—NH—} wherein } is point of        attachment with group W;    -   W is —C(O)—NH—(CH₂)₄—NH—], wherein ] is point of attachment of        group Y;    -   Y is —C(O)—(CH₂)₂—CH(COOH)NH—, wherein — is point of attachment        with the group Z;    -   Z is —C(O)—(CH₂)_(n)—COOH or —C(O)—(CH₂)_(n)—CH₃ wherein n is an        integer from 14 to 20.

In some embodiments, X21 is Lys wherein the side chain amino (ε amino)group of Lys is acylated with a moiety:{-Q-T-U—W—Y—Z,

-   -   wherein Z is —C(O)—(CH₂)_(n)—COOH wherein n is integer 16.

In some embodiments, X21 is Lys wherein the side chain amino (ε amino)group of Lys is acylated with a moiety:{-Q-T-U—W—Y—Z,

-   -   wherein Z is —C(O)—(CH₂)_(n)—CH₃ wherein n is integer 14.

In some embodiments, the present disclosure provides a polypeptidecomprising the amino acid sequence:

-   -   H-X2-X3-X4-G-T-F-T-S-D-V-S-S-Y-L-X16-G-Q-A-A-X21-E-F-X24-A-W-L-V-R-G-R-G-X33-X34    -   wherein X2 is Aib;    -   X3 is absent;    -   X4 is Glu;    -   X16 is Glu;    -   X24 is Ile;    -   X33 is Leu;    -   X34 is absent and    -   X21 is Lys wherein the side chain amino (ε amino) group of Lys        is acylated with a moiety:        {-Q-T-U—W—Y—Z    -   wherein Q and T are absent;    -   U is —C(O)—CH₂—O—(CH₂)₂—O—(CH₂)₂—NH—} wherein } is point of        attachment with group W;    -   W is

-   -    wherein ] is point of attachment with group Y;    -   Y is —C(O)—(CH₂)₂—CH(COOH)NH—, wherein — is point of attachment        with the group Z; and    -   Z is —C(O)—(CH₂)_(n)—COOH wherein n is integer 16.

In some embodiments, the present disclosure provides a polypeptidecomprising the amino acid sequence:

-   -   H-X2-X3-X4-G-T-F-T-S-D-V-S-S-Y-L-X16-G-Q-A-A-X21-E-F-X24-A-W-L-V-R-G-R-G-X33-X34    -   wherein X2 is Ser, Ser(OMe), D-Ser, D-Ser(OMe);    -   X3 is absent;    -   X4 is Glu;    -   X16 is Glu;    -   X24 is Ile;    -   X33 is Leu;    -   X34 is absent and    -   X21 is Lys wherein the side chain amino (ε amino) group of Lys        is acylated with a moiety:        {-Q-T-U—W—Y—Z    -   wherein Q and T are absent;    -   U is —C(O)—CH₂—O—(CH₂)₂—O—(CH₂)₂—NH—} wherein } is point of        attachment with group W;    -   W is —C(O)—CH₂—O—(CH₂)₂—O—(CH₂)₂—NH—, —C(O)—NH—(CH₂)₃₋₄—NH—],        —C(O)—C(CH₃)₂—NH—], wherein ] is point of attachment with group        Y;    -   Y is —C(O)—(CH₂)₂—CH(COOH)NH—, wherein — is point of attachment        with the group Z; and    -   Z is —C(O)—(CH₂)_(n)—COOH or —C(O)—(CH₂)_(n)—CH₃ wherein n is an        integer from 14 to 20.

In some embodiments, X21 is Lys wherein the side chain amino (ε amino)group of Lys is acylated with a moiety:{-Q-T-U—W—Y—Z,

-   -   wherein W is —C(O)—NH—(CH₂)₃₋₄—NH—].

In some embodiments, X21 is Lys wherein the side chain amino (ε amino)group of Lys is acylated with a moiety:{-Q-T-U—W—Y—Z,

-   -   wherein W is —C(O)—CH₂—O—(CH₂)₂—O—(CH₂)₂—NH—;

In some embodiments, X21 is Lys wherein the side chain amino (ε amino)group of Lys is acylated with a moiety:{-Q-T-U—W—Y—Z,

-   -   wherein W is —C(O)—C(CH₃)₂—NH—]

In some embodiments, X21 is lipid modified Lys wherein the side chainamino (ε amino) group of Lys is acylated with a moiety{-Q-T-U—W—Y—Z,

-   -   which is represented by the moieties provided in Table 1.

The present invention also provides a polypeptide comprising the aminoacid sequence:

-   -   H-X2-X3-X4-G-T-F-T-S-D-V-S-S-Y-L-X16-G-Q-A-A-X21-E-F-X24-A-W-L-V-R-G-R-G-X33-X34    -   wherein X2 is Ser, Ser(Me), Aib, Ala, Gly, Val, Leu, Ile,        (1-amino C₃₋₈ cycloalkyl) carboxylic acid, Trp or Thr;    -   X3 is absent or Gln;    -   X4 is Gln or Glu;    -   X16 is Glu or Asp;    -   X24 is Ile or Val or Leu;    -   X33 Leu, Ile, Tyr or absent;    -   X34 is Ser, Lys, Ala or absent and    -   X21 is Lys wherein the side chain amino (ε amino) group of Lys        is acylated with a moiety:        {-Q-T-U—W—Y—Z    -   wherein Q, T, U and W are absent or selected from a group        consisting of —C(O)-M-NH— wherein M is a C₅₋₁₀ alk chain        interrupted with 1, 2 or 3 oxygen atoms, —C(O)—N(R₁)—C₂₋₅        alk-N(R₁)—, —C(O)—C(CH₃)₂—N(R₂)— and

-   -   wherein R₁, R₂ or R₃ is independently selected from hydrogen or        C₁₋₃ alkyl;    -   provided that:        -   i. at least one of Q, T, U and W is always present and,        -   ii. at least one of Q, T, U and W is always selected from            groups C(O)—N(R₁)—C₂₋₅ alk-N(R₁)—, —C(O)—C(CH₃)₂—N(R₂)— and

-   -   Y is either absent or —C(O)—(CH₂)_(m)—CH(COOH)NH— wherein m is        an integer selected from 1 to 2 and — is point of attachment        with the group Z;    -   Z is —C(O)—(CH₂)_(n)—COOH or —C(O)—(CH₂)_(n)—CH₃ wherein n is an        integer from 10 to 20.

Unless stated otherwise, the specification intends to cover both L and Disomers of the amino acids in the sequence.

Ser(OMe) as described herein in the specification is amino acid serinewith its hydroxyl group methylated and has following structure.

In another aspect, the present invention provides a polypeptidecomprising the amino acid sequence:

-   -   H-X2-X3-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-X21-E-F-I-A-W-L-V-R-G-R-G-X33-X34    -   wherein X2 is Ser, Ser(OMe), Aib, Ala, Gly, Val, Leu, Ile,        (1-amino C₃₋₈ cycloalkyl) carboxylic acid, Trp or Thr;    -   X3 is absent or Gln;    -   X4 is Glu;    -   X33 is Leu or Ile;    -   X34 is absent or Ala and    -   X21 is Lys wherein the side chain amino (ε amino) group of Lys        is acylated with a moiety with following structure:        {-Q-T-U—W—Y—Z.        The moiety        {-Q-T-U—W—Y—Z    -   is also referred as the fatty acid side chain and protracting        moiety in the specification.

In another aspect the present invention provides polypeptide comprisingthe amino acid sequence:

-   -   H-X2-X3-X4-G-T-F-T-S-D-V-S-S-Y-L-X16-G-Q-A-A-X21-E-F-X24-A-W-L-V-R-G-R-G-X33-X34    -   wherein X2 is Ser, Ser(OMe), Aib, Ala, Gly, Val, Leu, Ile,        (1-amino C₃₋₈ cycloalkyl) carboxylic acid, Trp or Thr;    -   X3 is absent or Gln;    -   X4 is Gln or Glu;    -   X16 is Glu or Asp;    -   X24 is Ile or Val or Leu;    -   X33 is Leu, Ile or Tyr;    -   X34 is absent or Ala and    -   X21 is Lys wherein the side chain amino (ε amino) group of Lys        is acylated with a moiety:        {-Q-T-U—W—Y—Z    -   wherein Q, T, U and W are absent or selected from a group        consisting of —C(O)-M-NH— wherein M is a C₅₋₁₀ alk chain        interrupted with 1, 2 or 3 oxygen atoms, —C(O)—NH—C₂₋₅ alk-NH—,        —C(O)—C(CH₃)₂—NH— and

-   -   provided that:        -   i. at least one of Q, T, U and W is always present and,        -   ii. at least one of Q, T, U and W is always selected from a            group consisting of —C(O)—NH—C₂₋₅ alk-NH—, —C(O)—C(CH₃)₂—NH—            and

The bond depicted as “—” is point of attachment with the group on theright hand side of the structure. For example when Q is —C(O)-M-NH—, theNH— group is attached to T.

In the group —C(O)-M-NH—, M is a C₅₋₁₀ alk chain interrupted with 1, 2or 3 oxygen atoms. The term “interrupted” means that between two carbonsof the alkyl chain an oxygen atom is inserted. The oxygen atom cannot beat the end of the chain (terminal position) and two oxygen atoms cannotbe present adjacent (peroxide linkage) to each other. The chain lengthis excluding the number of oxygen atoms i.e. if a C₅ alkyl chain isinterrupted with two oxygen atoms then the total chain length will be oftotal 7 atoms.

In another preferred embodiment, the group —C(O)—NH—C₂₋₅ alk-NH— is—C(O)—NH—C₃H₇—NH—. In another preferred embodiment, the group—C(O)—NH—C₂₋₅ alk-NH— is —C(O)—NH—C₄H₈—NH—.

In an embodiment, the amino acid at X2 is selected from Ser, Ser(OMe),Ala or Aib.

In another embodiment, X3 is absent.

In another embodiment, X3 is absent and X4 is Gln.

In a preferred embodiment, X33 is Leu or Ile and X34 is Ala.

In another preferred embodiment, X33 is Leu or Ile and X34 is absent.

In another embodiment X21 is Lys wherein the side chain amino (ε amino)group of Lys is acylated with a moiety:{-Q-T-U—W—Y—Z.

In another embodiment, W is absent and T and W are —C(O)-M-NH— and U isselected from a group consisting of —C(O)-M-NH—, —C(O)—NH—C₂₋₅ alk-NH—,—C(O)—C(CH₃)₂—NH— and

In another embodiment, T is —C(O)-M-NH—; U is selected from a groupconsisting of —C(O)-M-NH—, —C(O)—NH—C₂₋₅ alk-NH—, —C(O)—C(CH₃)₂—NH— and

-   -   and Q and W are absent.

In another embodiment, X21 is lipid modified Lys wherein the side chainamino (ε amino) group of Lys is acylated with a moiety:{-Q-T-U—W—Y—Z

-   -   wherein Q is absent; T is selected from a group consisting of        —C(O)—NH—C₂₋₅ alk-NH—, —C(O)—C(CH₃)₂—NH— and

-   -    and    -   U and W are —C(O)-M-NH—.

The protracting moieties of the present invention have bonds which areless susceptible to cleavage by the biological enzymes thus affordingcompounds with long duration of action. For example, when Q, T, U or Wis selected from —C(O)—NH—C₂₋₅ alk-NH— or

it forms a —NH—C(O)—NH— (urea) linkage which is relatively difficult toget cleaved by the enzymes when compared to simple amide bonds.Similarly, when Q, T, U or W is —C(O)—C(CH₃)₂—NH— (Aib) it forms anamide linkage which is relatively more stable.

Accordingly, in an embodiment, X21 is lipid modified Lys wherein theside chain amino (E amino) group of Lys is acylated with a moiety:{-Q-T-U—W—Y—Z

-   -   wherein    -   i. at least one of Q, T, U and W is always present and,    -   ii. at least one of Q, T, U and W is always selected from groups        —C(O)—NH—C₂₋₅ alk-NH—, —C(O)—C(CH₃)₂—NH— and

In another embodiment, Q and T are —C(O)-M-NH— and U is selected from agroup consisting of —C(O)—NH—C₂₋₅ alk-NH—, —C(O)—C(CH₃)₂—NH— and

-   -    and W is absent.

In another embodiment, Q is —C(O)-M-NH—; T is selected from a groupconsisting of —C(O)—NH—C₂₋₅ alk-NH—, —C(O)—C(CH₃)₂—NH— and

-   -    and    -   U and W are absent.

In another embodiment, Q is selected from a group consisting of—C(O)—NH—C₂₋₅ alk-NH—, —C(O)—C(CH₃)₂—NH— and

-   -   T and U are —C(O)-M-NH— and W is absent.

In a preferred embodiment, —C(O)-M-NH— group is —C(O)—CH₂O—C₂H₅OC₂H₅NH—.

In another embodiment, wherein Q is —C(O)—CH₂O—C₂H₅OC₂H₅NH—; T is—C(O)—NH—(CH₂)₃₋₄NH— and U and W are absent.

In another embodiment, Q is —C(O)—CH₂O—C₂H₅OC₂H₅NH—; T is

-   -    and U and W are absent.

In another embodiment, Q is —C(O)—CH₂O—C₂H₅OC₂H₅NH—; T is—C(O)—C(CH₃)₂—NH— and U and W are absent.

In another embodiment, Q and U are —C(O)—CH₂O—C₂H₅OC₂H₅NH—; T is—C(O)—C(CH₃)₂—NH— and W is absent.

In another embodiment, X21 is lipid modified Lys wherein the side chainamino (ε amino) group of Lys is acylated with a moiety{-Q-T-U—W—Y—Z

-   -   which is represented by the moieties provided in Table 1.

TABLE 1 Representative moieties for group {-Q-T-U-W-Y-Z DesignationMoiety Moiety A

Moiety B

Moiety C

Moiety D

Moiety E

Moiety F

In another embodiment, the present disclosure provides a polypeptideaccording to any one of the preceding embodiments, which is selectedfrom the peptides provided in Table 2:

TABLE 2 Representative polypeptide compounds of present disclosure Comp.Seq # Structure* ID  1

SEQ ID NO: 05  2

SEQ ID NO: 06  3

SEQ ID NO: 07  4

SEQ ID NO: 08  5

SEQ ID NO: 09  6

SEQ ID NO: 10  7

SEQ ID NO: 05  8

SEQ ID NO: 11  9

SEQ ID NO: 12 10

SEQ ID NO: 13 11

SEQ ID NO: 14 12

SEQ ID NO: 10 13

SEQ ID NO: 05 14

SEQ ID NO: 09 15

SEQ ID NO: 11 16

SEQ ID NO: 05 17

SEQ ID NO: 05 18

SEQ ID NO: 05 *Unless stated otherwise, all the amino acids in thestructures have L configuration at α position. D when used as a prefixto amino acid in the sequence denotes the D configuration of the aminoacid. For example (DSer) denotes that Serine amino acid in the sequencehas D-configuration.

Unless stated otherwise, the disclosure intends to cover both L and Disomers of the amino acids in the sequences.

Ser(OMe) as described herein in the disclosure is amino acid serine withits hydroxyl group methylated and has following structure.

The polypeptide sequences mentioned in the disclosure are represented bythe single letter code of the amino acids as approved by IUPAC.

Q, T, U, W, Y and Z as used herein to define the acylating moiety of theembodiments of the present disclosure are different than the singleletter code of the amino acid used to denote the polypeptide sequence.

The polypeptides of the present disclosure surprisingly showedsignificant reduction in the blood glucose when subjected to an oralglucose tolerance test (OGTT) in SD rats. The percentage reduction ofblood glucose in SD rats when challenged with oral glucose wassignificantly lower than the corresponding polypeptides which lacked theadditional Leu or Ile at X33 position.

The present invention is further illustrated in detail with reference tothe following examples. It is desired that the example be considered inall respect as illustrative and are not intended to limit the scope ofthe claimed invention.

EXAMPLES

General Methods of Preparation:

The polypeptide compound of the present disclosure can be prepared bymethods described herein below. The process involves two steps,involving preparation of the parent linear peptide and subsequentattachment of fatty acid chain to the parent peptide.

The peptides described herein may be prepared by chemical synthesisusing solid-phase techniques such as those described in G. Barany and R.B. Merrifield, “The Peptides: Analysis, Synthesis, Biology”; Volume2—“Special Methods in Peptide Synthesis, Part A”, pp. 3-284, E. Grossand J. Meienhofer, Eds., Academic Press, New York, 1980; and in J. M.Stewart and J. D. Young, “Solid-Phase Peptide Synthesis”, 2nd Ed.,Pierce Chemical Co., Rockford, Ill., 1984. The desired strategy is basedon the Fmoc (9-Fluorenylmethyl-oxycarbonyl) group for temporaryprotection of the α-amino group, in combination with protecting groupssuch as tert-butyl (-tBu), tert-butyloxycarbonyl (-Boc), trityl (-Trt)groups for temporary protection of the amino acid side chains (see forexample E. Atherton and R. C. Sheppard, “The FluorenylmethoxycarbonylAmino Protecting Group”, in “The Peptides: Analysis, Synthesis,Biology”; Volume 9-“Special Methods in Peptide Synthesis, Part C”, pp.1-38, S. Undenfriend and J. Meienhofer, Eds., Academic Press, San Diego,1987).

The peptides can be synthesized in a stepwise manner on an insolublepolymer support (also referred to as “resin”) starting from theC-terminus of the peptide. A synthesis is begun by appending theC-terminal amino acid of the peptide to the resin through formation ofan amide or ester linkage. This allows the eventual release of theresulting peptide as a C-terminal amide or carboxylic acid,respectively.

The C-terminal amino acid and all other amino acids used in thesynthesis are required to have their α-amino groups and side chainfunctionalities (if present) differentially protected such that theα-amino protecting group may be selectively removed during thesynthesis. The coupling of an amino acid is performed by activation ofits carboxyl group as an active ester and reaction thereof with theunblocked a-amino group of the N-terminal amino acid appended to theresin. The sequence of a-amino group deprotection and coupling isrepeated until the entire peptide sequence is assembled. The peptide isthen released from the resin with concomitant deprotection of the sidechain functionalities, usually in the presence of appropriate scavengersto limit side reactions. The resulting peptide is finally purified byreverse phase HPLC.

The parent peptide can then be coupled to the fatty acid chain bycoupling the activated fatty acid chain with the parent peptide. Thefatty acid chain may be made by methods well known in organic chemistry.For example, the fatty acid chain can be made using solid phasesynthetic methods which enables preparation of linear fatty acid chains.

The linear peptides synthesized were purified by preparative HPLCprocedure as outlined below:

-   -   Preparative HPLC: WATERS 2555 Quaternary gradient module (Max        Total Flow: 300 mL/min, Max Pressure: 3000 psi) or    -   Shimadzu LC-8A (Max Total Flow: 150 mL: Max Pressure: 20 Mpa)    -   Column: C18, 10μ    -   Flow: 75 mL/min    -   Mobile Phase: For first purification        -   Mobile Phase A: pH 7.5 Phosphate buffer        -   Mobile Phase B: Acetonitrile        -   Gradient: 10 to 40% Mobile Phase-B in 300 min.        -   For second purification:        -   Mobile Phase A: 1% Acetic acid in water        -   Mobile Phase B: 1% Acetic acid in Acetonitrile:n-Propanol            (50:50)        -   Gradient: 15 to 45% Mobile phase-B in 300 min

The final compounds of the present disclosure were purified bypreparative HPLC procedure as outlined below:

-   -   Preparative HPLC: WATERS 2555 Quaternary gradient module (Max        Total Flow: 300 mL/min, Max Pressure: 3000 psi) or    -   Shimadzu LC-8A (Max Total Flow: 150 mL, Max Pressure: 20 Mpa)    -   Column: C18, 10μ    -   Flow: 75 mL/min

Mobile Phase:

For first purification For second purification Mobile Phase A pH 7.5Phosphate buffer 1% Acetic acid in water Mobile Phase B Acetonitrile 1%Acetic acid in ) Acetonitrile:n-Propanol (50:50 Gradient 10 to 40%Mobile 15 to 45 % Mobile Phase-B Phase-B in 300 min in 300 min

The purity of the compounds of the present disclosure was analysed byRP-HPLC method as outlined below:

HPLC Method B1:

-   -   Column: YMC Pack-Ph (4.6 mm×150 mm 3μ)    -   Eluent: Mobile Phase A: 0.1% Trifluroacetic acid in Water    -   Mobile phase B: 0.1% Trifluroacetic acid in Acetonitrile    -   Flow rate: 1.5 mL/min    -   Detection: UV detection at 210 nm    -   Column Temperature: 50° C.    -   Run Time: 50 min.    -   Gradient:

Time Mobile Phase A % Mobile Phase B % 0.01 90 10 35.0 20 80 40.0 20 8041.0 90 10 50.0 90 10HPLC Method B2:

-   -   Column: YMC-Pack Pro C18 (4 mm×250 mm, 3μ)    -   Eluent: Mobile Phase A: Buffer: Acetonitrile (900:100)    -   Mobile phase B: Buffer: Acetonitrile (300:700    -   Buffer: Potassium dihydrogen orthophosphate in water, pH        adjusted to 3.0±0.1 with    -   orthophosphoric acid    -   Flow rate: 1.0 mL/min    -   Detection: UV detection at 210 nm    -   Column Temperature: 50° C.    -   Sample Tray temperature: 8° C.    -   Run Time: 38 min.

Time Mobile Phase A % Mobile Phase B % 0 100 0 5 100 0 30 0 100 32 0 10032.1 100 0 38 100 0HPLC Method B3:

-   -   Column: Waters X-Select CSH-C18 (150 mm×4.6 mm; 2.5μ)    -   Eluent: Mobile Phase A: Buffer: Acetonitrile (900:100)    -   Mobile phase B: Buffer: Acetonitrile (300:700    -   Buffer: Potassium dihydrogen orthophosphate in water, pH        adjusted to 1.5±0.1 with orthophosphoric acid    -   Flow rate: 0.9 mL/min    -   Detection: UV detection at 210 nm    -   Column Temperature: 40° C.    -   Sample Tray temperature: 5° C.    -   Run Time: 100 min.

Time Mobile Phase A Mobile Phase B 0 50 50 40 43 57 55 43 57 90 0 100 9150 50 100 50 50

The compounds of the present disclosure were analyzed by the LCMS asoutlined below:

Mass spectra were recorded on LCMS using Waters Acquity® QDa®, WatersMicromass Quattro Micro API or Thermo scientific LCQ Fleet™. The testsolution was prepared by dissolving a suitable quantity of analyte indiluent with a final concentration from 1 μg/ml to 50 μg/ml depending onthe ionization of analyte. The test solution was infused at a rate ofabout 10 μl to 50 μl per minute into LCMS for 1 min and mass spectrawere recorded in Electro Spray Ionization (ESI) positive or negativemode and in an appropriate mass range.

Example 1: Preparation of Activated Fatty Acid Side Chains 1.Preparation of18-[[(1S)-1-carboxy-4-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrolidin-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-4-oxo-butyl]amino]-18-oxo-octadecanoicacid (Moiety A-OSu, Intermediate-3)

The activated fatty acid side chain, Moiety A-OSu was prepared by solidphase synthesis using 2-chlorotrityl chloride resin as schematicallyrepresented in FIG. 1A. 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid(Intermediate-1) was attached to 2-chlorotrityl chloride resin inpresence of N,N′-di-isopropylethylamine (DIPEA) which yielded2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid-2-Cl-Trt-Resin. Theintermediate-1 can be prepared by coupling of 2-[2-(2-aminoethoxy)-ethoxy] acetic acid with Fmoc N-hydroxysuccinimide ester.Alternatively, intermediate-1 is available commercially and can beprocured as such. The Fmoc protecting group was removed by selectivede-blocking of amino group of 2-[2-(2-Fmoc-aminoethoxy)ethoxy] aceticacid-2-Cl-Trt-Resin using piperidine and the free amino group was thencoupled to 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid using1-hydroxybenztriazole (HOBt) and N,N′-di-isopropylcarbodiimide (DIPC)which yielded 2-[2-[2-[[2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin. The Fmoc group was thenremoved by selective de-blocking of amino group of2-[2-[2-[[2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin using piperidine and thefree amino group was then coupled to Fmoc-Glu-OtBu using HOBt and DIPCto obtain2-[2-[2-[[2-[2-[2-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin. The resultant2-[2-[[2-[2-[2-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin wasselectively deblocked using piperidine and then coupled withoctadecanedioic acid mono tert-butyl ester to give intermediate-2 namely[2-[2-[2-[[2-[2-[2-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid]-2-Cl-Trt-Resin. The intermediate 2 was then cleaved from2-Cl-Trt-Resin using trifluoroethanol:DCM (1:1). The resultant compoundwas then reacted with N-hydroxysuccinimide (HOSu) in presence ofisobutyl chloroformate (IBCF) and N-methylmorpholine (NMM) followed byde-protection with trifluoroacetic acid to yield the title compound(Moiety A-OSu, intermediate-3). The whole process can also be depictedas schematically represented in FIG. 1B.

2. Preparation of N-Palmitoyl-L-γ-Glutamyl Succinimide Ester (MoietyB-OSu)

L-Glutamic acid alpha-tert-butyl ester (H-Glu-OtBu) was reacted withpalmitic acid in presence of IBCF and NMM to yieldCH₃—(CH₂)₁₄—C(O)-Glu-OtBu, which was then reacted with HOSu in thepresence of IBCF and NMM to yield CH₃—(CH₂)₁₄—C(O)-Glu(OSu)-OtBu, whichwas then de-protected with trifluoroacetic acid to yield Moiety B-OSu.

3. Preparation of18-[[(1S)-1-carboxy-4-[4-[2-[2-[2-(2,5-dioxopyrrolidin-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylcarbamoylamino]butylamino]-4-oxo-butyl]amino]-18-oxo-octadecanoicAcid (Moiety C-OSu)

The activated fatty acid side chain, Moiety C-OSu was prepared usingsolid phase synthesis using 2-chlorotrityl chloride resin asschematically represented in FIG. 2 .2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid was attached to2-chlorotrityl chloride resin in presence of DIPEA to yield2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid-2-Cl-Trt-Resin. The Fmocprotecting group was removed by selective de-blocking of amino groupusing piperidine and the free amino group was then activated usingp-nitrophenylchlroformate in THF and DIPEA followed by reaction withFmoc-amino butylamine hydrochloride salt in THF: DMAc and DIPEA, whichyielded 2-[2-[2-(4-Fmoc-aminobutylcarbamoylamino)ethoxy]ethoxy]aceticacid-2-Cl-Trt-Resin. The Fmoc group was removed by selective de-blockingusing piperidine and the free amino group was then coupled toFmoc-Glu-OtBu using of HOBt and DIPC, which yielded2-[2-[2-[4-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]butylcarbamoylamino]ethoxy]ethoxy]aceticacid-2-Cl-Trt-Resin. The resultant2-[2-[2-[4-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]-butylcarbamoylamino]ethoxy]ethoxy]aceticacid-2-Cl-Trt-Resin was selectively deblocked using piperidine and thencoupled with octadecanedioic acid mono tert-butyl ester to giveintermediate2-[2-[2-[4-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]butylcarbamoylamino]ethoxy]ethoxy]aceticacid-2-Cl-Trt-Resin. The intermediate was then cleaved from2-Cl-Trt-Resin using trifluoroethanol:DCM (1:1) to obtain2-[2-[2-[4-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]butylcarbamoylamino]ethoxy]ethoxy]aceticacid (LCMS=m/z: 814.56 (M+H⁺)). The resultant compound was then reactedwith HOSu in presence of dicyclohexyl carbodiimide (DCC) to yieldsuccinimide protected intermediate, which was de-protected withtrifluoroacetic acid to yield the title compound (Moiety C-OSu).

4. Preparation of18-[[(1S)-1-carboxy-4-[[2-[2-[2-[2-(2,5-dioxopyrrolidin-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-1,1-dimethyl-2-oxo-ethyl]amino]-4-oxo-butyl]amino]-18-oxo-octadecanoicAcid (Moiety D-OSu)

The fatty acid side chain was prepared using solid phase synthesis using2-chlorotrityl chloride resin as schematically represented in FIG. 3 .2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid was attached to2-chlorotrityl chloride resin in presence of DIPEA to yield2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid-2-Cl-Trt-Resin. The Fmocprotecting group was removed by selective de-blocking of amino groupusing piperidine followed by coupling with Fmoc-Aib-OH in THF: DMAcusing DIPC and HOBt which yielded2-[2-[2-[(2-Fmoc-amino-2-methyl-propanoyl)amino]ethoxy]ethoxy]aceticacid-2-Cl-Trt-Resin. The Fmoc group was removed by selective de-blockingusing piperidine and the free amino group was coupled with Fmoc-Glu-OtBuusing HOBt and DIPC to yield2-[2-[2-[[2-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]-2-methyl-propanoyl]amino]ethoxy]ethoxy]aceticacid-2-Cl-Trt-Resin. The Fmoc group of the resultant compound wasselectively de-blocked using piperidine and the free amino group wasthen coupled with octadecanedioic acid mono tert butyl ester to give2-[2-[2-[[2-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]-2-methyl-propanoyl]-amino]ethoxy]ethoxy]aceticacid-2-Cl-Trt-Resin. The intermediate was then cleaved from2-Cl-Trt-Resin using trifluoroethanol:DCM (1:1) to obtain2-[2-[2-[[2-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]-2-methyl-propanoyl]amino]ethoxy]ethoxy]aceticacid (LCMS=m/z: 786.39 (M+H⁺)). The resultant compound was then reactedwith HOSu in presence of DCC to yield succinimide protectedintermediate, which was de-protected with trifluoroacetic acid to yieldthe title compound (Moiety D-OSu).

5. Preparation of18-[[(1S)-1-carboxy-4-[3-[2-[2-[2-(2,5-dioxopyrrolidin-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylcarbamoylamino]propylamino]-4-oxo-butyl]amino]-18-oxo-octadecanoicAcid (Moiety E-OSu)

The fatty acid side chain was prepared using solid phase synthesis using2-chlorotrityl chloride resin as schematically represented in FIG. 4 .2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid was attached to2-chlorotrityl chloride resin in presence of DIPEA to yield2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid-2-Cl-Trt-Resin. The Fmocprotecting group was removed by selective de-blocking of amino groupusing piperidine and the free amino group was then activated usingp-nitrophenylchloroformate in THF and DIPEA followed by reaction with1,3-diaminopropane in THF: DMAc in presence of DIPEA using HOBt to formNH₂—(CH₂)₃—NH—C(O)-{(2-(2-amino-ethoxy)-ethoxy}-aceticacid-2-Cl-Trt-Resin. The free amino group was then coupled toFmoc-Glu-OtBu using HOBt and DIPC, which yielded2-[2-[2-[3-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]propylcarbamoylamino]ethoxy]ethoxy]aceticacid-2-Cl-Trt-Resin. The resultant2-[2-[2-[3-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]propylcarbamoylamino]ethoxy]-ethoxy]aceticacid-2-Cl-Trt-Resin was selectively deblocked using piperidine and thencoupled with octadecanedioic acid mono tert butyl ester to give2-[2-[2-[3-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]-propylcarbamoylamino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin. Theintermediate was then cleaved from 2-Cl-Trt-Resin usingtrifluoroethanol:DCM (1:1) to obtain2-[2-[2-[3-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]-propylcarbamoylamino]ethoxy]ethoxy]aceticacid (LCMS=m/z: 801.41 (M+H⁺)). The resultant compound was then reactedwith HOSu in presence of dicyclohexyl carbodiimide (DCC) to yieldsuccinimide protected intermediate, which was de-protected withtrifluoroacetic acid to yield the title compound (Moiety E-OSu).

6. Preparation of18-[[(1S)-1-carboxy-4-[4-[2-[2-[2-(2,5-dioxopyrrolidin-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylcarbamoylamino]-1-piperidyl]-4-oxo-butyl]amino]-18-oxo-octadecanoicAcid (Moiety F-OSu)

The fatty acid side chain was prepared using solid phase synthesis using2-chlorotrityl chloride resin as schematically represented in FIG. 5 .2-[2-(2-Fmoc-amino ethoxy)ethoxy]acetic acid was attached to2-chlorotrityl chloride resin in presence of DIPEA to yield2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid-2-Cl-Trt-Resin. The Fmocprotecting group was removed by selective de-blocking of amino groupusing piperidine and the free amino group was then activated usingp-nitrophenylchloroformate in THF and DIPEA followed by reaction with4-amino-Boc-piperidine in THF: DMAc in presence of DIPEA using HOBtwhich yielded (2-[2-[2-(4-Boc-piperidylcarbamoylamino)ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin. The resultant compound oncleavage using trifluoro acetic acid yielded2-[2-[2-(4-piperidylcarbamoylamino)ethoxy]ethoxy]acetic acid which onfurther reaction with Fmoc-OSu in presence of triethylamine (TEA)yielded 2-[2-[2-(4-Fmoc-piperidylcarbamoylamino)ethoxy]ethoxy]aceticacid. The obtained compound was then further attached to 2-chlorotritylchloride resin in presence of DIPEA to yield2-[2-[2-(4-Fmoc-piperidylcarbamoylamino)ethoxy]ethoxy]aceticacid-2-Cl-Trt-Resin. The Fmoc group was removed by selective de-blockingusing piperidine and the free amino group was then coupled withFmoc-Glu-OtBu using HOBt and DIPC, which yielded2-[2-[2-[[1-[(4S)-4-amino-5-tert-butoxy-5-oxo-pentanoyl]-4-piperidyl]carbamoylamino]ethoxy]ethoxy]aceticacid-2-Cl-Trt-Resin. The resultant compound was selectively de-blockedusing piperidine and was then coupled with octadecanedioic acid monotert-butyl ester to give2-[2-[2-[[1-[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]-4-piperidyl]carbamoylamino]ethoxy]ethoxy]aceticacid-2-Cl-Trt-Resin. Intermediate was then cleaved from 2-Cl-Trt-Resinusing trifluoroethanol:DCM (1:1) to obtain2-[2-[2-[[1-[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]-4-piperidyl]carbamoylamino]ethoxy]ethoxy]aceticacid (LCMS=m/z: 827.40 (M+H⁺)). The resultant compound was then reactedwith HOSu in presence of DCC to yield succinimide protectedintermediate, which was de-protected using trifluoroacetic acid to yieldthe title compound, Moiety F-OSu.

Example 2: Synthesis of Compound 1N-ε²⁶-[2-(2-[2-(2-[2-(2-[4-(17-Carboxyheptadecanoylamino)-4(S)carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][(Aib⁸, Arg³⁴, Leu³⁸ GLP-1(7-38)Peptide

Part A. Synthesis of the Parent Linear Peptide Aib⁸, Arg³⁴, Leu³⁸GLP-1(7-38)

The parent peptide was synthesized by solid-phase method. The startingresin used for synthesis was Wang resin. Fmoc protected Leucine was usedfor coupling with the Wang resin. The coupling was performed by usingdiisopropylcarbodiimide, N-hydroxybenzotriazole (DIC-HOBt) as couplingreagent in presence of 4-dimethylaminopyridine (DMAP) which yieldedFmoc-Leu-Wang Resin. Selective de-blocking of amino group ofFmoc-Leu-Wang Resin using piperidine followed by coupling withFmoc-Gly-OH using HOBt and DIPC yielded Fmoc-Gly-Leu-Wang Resin. Thiscompletes one cycle. Acetic anhydride and diisopropylethylamine/pyridine was used to terminate the uncoupled amino groups at everyamino acid coupling.

The above 2 steps, i.e., selective deblocking of Fmoc-protection ofamino acid attached to the resin and coupling of next amino acid residuein sequence with Fmoc-protected amino group were repeated for remaining30 amino acid residues. The selective deblocking, i.e., deprotection ofFmoc group was done using piperidine and coupling with next Fmocprotected amino acid was done using HOBt/DIPC. The side chain of theFmoc-protected amino acids were protected orthogonally, e.g., hydroxylgroup of Serine, Tyrosine or Threonine were protected withtert-butyl(-tBu) group, amino and guanido group of Lysine and Argininewere protected with tert-butyloxycarbonyl (-Boc) and2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (-Pbf) grouprespectively, the imidazole of histidine was protected with trityl(-Trt) and carboxylic acid groups of aspartic acid or glutamic acid wereprotected with -tBu group. The above mentioned two steps, i.e.,selective deblocking and then coupling with next Fmoc protected aminoacid were performed to getFmoc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-Ile-Ala-Trp-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Leu-resin.

De-blocking ofFmoc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-Ile-Ala-Trp-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Leu-Resin using piperidine followed by cleavage and de-protection usingtrifluoroacetic acid with ethane-1,2-dithiol resulted in crudeH-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-Leu-OH(Aib⁸, Arg³⁴, Leu³⁸ GLP-1 (7-38) peptide) which was purified by HPLC.

Part B:

Grafting of activated fatty acid chain, Moiety A-OSu over purified(Linear Peptide)H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-Leu-OHobtained in Part A, in acetonitrile at pH about 10 resulted in crudetitle peptide which was purified by preparative HPLC. Thecharacterization of the compounds is provided in Table 3.

Example 3: Preparation of Compound 2, 3, 5, 9, 10, and 12

The linear peptides of the compounds 2, 3, 5, 9, 10 and 12 were preparedby solid phase method as per the analogous process given for Example 1,Part A. Grafting of activated fatty acid chain, Moiety A-OSu byfollowing the process of Example 1, Part B over the respective linearpeptides afforded compound 2, 3, 5, 9, 10 and 12.

Example 4: Preparation of Compound 4 and 11

The linear peptides of the Compound 4 and 11 were prepared by solidphase method as per the analogous process given for Example 2, Part Aexcept here Fmoc protected D-Leucine was first coupled with Wang resinand then sequentially other amino acids were coupled. Grafting ofactivated fatty acid chain, Moiety A-OSu over the respective linearpeptide by following the process of Example 2, Part B afforded theCompound 4 and 11.

Example 5: Preparation of Compound 8

The linear peptide was prepared by solid phase method as per theanalogous process given for Example 2, Part A except here Fmoc protectedIsoleucine was first coupled with Wang resin and then sequentially otheramino acids were coupled. The grafting of activated fatty acid chain,Moiety A-OSu over the linear peptide by following the process of Example2, Part B afforded the Compound 8.

Example 6: Preparation of Compound 6

The linear peptide was prepared by solid phase method as per theanalogous process given for Example 2, Part A. The grafting of activatedfatty acid chain, Moiety B-OSu over the linear peptide by followinganalogous process of Example 2, Part B afforded the Compound 6.

Example 7: Preparation of Compound 7

The grafting of activated fatty acid chain, Moiety B-OSu over the linearpeptide of the Example 2, Part A, by following analogous process ofExample 2, Part B afforded the Compound 7.

Example 8: Preparation of Compound 13

The grafting of activated fatty acid chain, Moiety C-OSu over the linearpeptide of the Example 1, Part A, by following analogous process ofExample 1, Part B afforded the Compound 13.

Example 9: Preparation of Compound 14

The linear peptide was prepared by solid phase method as per theanalogous process given for Example 2, Part A. The grafting of activatedfatty acid chain, Moiety C-OSu over the linear peptide by followinganalogous process of Example 2, Part B afforded the Compound 14.

Example 10: Preparation of Compound 15

The linear peptide was prepared by solid phase method as per theanalogous process given for Example 2, Part A starting with Fmocprotected Isoleucine was first coupled with Wang resin and thensequentially other amino acids were coupled. The grafting of activatedfatty acid chain, Moiety C-OSu over the linear peptide by followinganalogous process of Example 2, Part B afforded the Compound 15.

Example 11: Preparation of Compound 16

The grafting of activated fatty acid chain, Moiety D-OSu over the linearpeptide of the Example 2, Part A, by following analogous process ofExample 2, Part B afforded the Compound 16.

Example 12: Preparation of Compound 17

The grafting of activated fatty acid chain, Moiety E-OSu over the linearpeptide of the Example 2, Part A by following analogous process ofExample 2, Part B afforded the Compound 17.

Example 13: Preparation of Compound 18

The grafting of activated fatty acid chain, Moiety F-OSu over the linearpeptide of the Example 2, Part A, by following analogous process ofExample 2, Part B afforded the Compound 18.

The characterization data of the synthesized compounds of the presentdisclosure are provided below in following Table 3.

TABLE 3 Characterization data of representative compounds of the presentdisclosure Comp. # LCMS Data HPLC Purity 1 m/z = 1057.52 (MH₄ ⁴⁺),Calculated Mass = 4226.05 98.32% (Method B2), RT = 24.85 min. 2 m/z =1061.74 (MH₄ ⁴⁺), Calculated Mass = 4242.93 99.02% (Method B1), RT =18.53 min. 3 m/z = 1087.65 (M − 4H)⁻⁴, Calculated Mass = 4354.63 98.12%(Method B1), RT = 18.48 min. 4 m/z = 1055.68 (M − 4H)⁻⁴, Calculated Mass= 4226.75 98.97% (Method B1), RT = 18.32 min. 5 m/z = 1057.88 (MH₄ ⁴⁺),Calculated Mass = 4227.49 96.75% (Method B1), RT = 17.09 min. 6 m/z =967.26 (MH₄ ⁴⁺), Calculated Mass: 3865.01 98.68% (Method B3), RT = 44.04min. 7 m/z = 968.53 (M − 4H)⁻⁴, Calculated Mass = 3878.15 97.39% (MethodB3), RT = 27.79 min. 8 m/z = 1057.72 (MH₄ ⁴⁺), Calculated Mass = 4226.8595.70% (Method B1), RT = 16.62 min. 9 m/z = 1061.67 (MH₄ ⁴⁺), CalculatedMass = 4242.65 95.15% (Method B1), RT = 16.48 min. 10 m/z = 1058.18 (MH₄⁴⁺), Calculated Mass = 4228.69 93.66% (Method B1), RT = 16.13 min. 11m/z = 1056.95 (MH₄ ⁴⁺), Calculated Mass: 4223.77 95.70% (Method B2), RT= 24.46 min 12 m/z = 1405.12 (MH₃ ³⁺), Calculated Mass: 4212.34 97.51%(Method B1), RT = 19.06 min. 13 m/z = 1049.59 (MH₄ ⁴⁺), Calculated Mass= 4194.33 96.01% (Method B2), RT = 25.16 min. 14 m/z = 1050.13 (MH₄ ⁴⁺),Calculated Mass = 4196.49 92.06% (Method B2), RT = 24.55 min. 15 m/z =1049.61 (MH₄ ⁴⁺), Calculated Mass = 4194.41 94.41% (Method B2), RT =24.82 min. 16 m/z = 1042.34 (MH₄ ⁴⁺), Calculated Mass = 4165.32 94.56%(Method B2), RT = 25.26 min. 17 m/z = 1046.18 (MH₄ ⁴⁺), Calculated Mass= 4180.72 94.33% (Method B2), RT = 25.17 min. 18 m/z = 1052.77 (MH₄ ⁴⁺),Calculated Mass = 4207.08 93.12% (Method B2), RT = 24.92 min.

Example 14: Oral Glucose Tolerance Test (OGTT) in Rats; SingleInjection; 1 mg/kg

Animals were divided into three groups—a normal control group, a testgroup and a third semaglutide group, with 4 animals in each group. Theanimals were fasted for 12 hours before initiation of OGTT. To the testgroup animals, the Compound 1 was injected subcutaneously at 1 mg/kgdose. To the semaglutide group, a dose of 1 mg/kg was injectedsubcutaneously. After 22 hrs, 166 hrs and 334 hrs of subcutaneousinjection of test drug or semaglutide, blood glucose was measured withblood glucose meter (time 0 measurements). All the animals were thengiven 2 g/kg of glucose solution orally. Blood glucose was measured at20, 40, 60, 90 and 120 minutes following glucose challenge. Body weightand food intake was recorded. Blood glucose data was analyzed using Twoway ANOVA followed by Bonferroni posttests using PRISM (Graph Padversion 5.03). Data of Blood glucose AUC_(0-120 min), was analyzed usingt test.

The polypeptides of the present disclosure have shown significantglucose lowering effect compared to the control group when studied inOral Glucose Tolerance Test (OGTT) in rats. For example, FIG. 6 provideschange in blood glucose level from time 0 to 120 min after 22 hrs and 46hrs in the test group administered Compound 1 and the semaglutidetreatment group. At 22 hours after single dosing, the Compound 1 showedstatistically significant reduction in blood glucose level with p<0.001vs normal control in ANOVA followed by Bonferroni's posttests. Theglucose lowering effect of the Compound 1 was superior to the glucoselowering effect observed with semaglutide (see FIG. 6A). The superiorityof glucose lowering effect of the Compound 1 was observed even after 46hours of subcutaneous administration (FIG. 6B). Further, both theCompound 1 and semaglutide showed statistically significant reduction infood intake as compared to control when observed on day 2 as well as onday 4 (see Table 4 & 5). The reduced food intake shown by Compound 1 onday 4 was greater than semaglutide (see Table 5). In terms of bodyweight reduction, only the test compound showed significant reduction onbody weight on day 4.

TABLE 4 Effect of treatment on food intake and body weight on Day 2 FoodConsumption (g) Body Weight (g) (day 0 to day 2) (Day 2) % Change MeanSD Mean SD vs Baseline Normal Control 33.0 4.7 480.6 15.6 8.89 Compound1 2.63*** 2.5 365.6 21.0 −9.33 Semaglutide 5.03*** 3.4 428.1 42.3 −7.65*p < 0.05, **p < 0.01, ***p < 0.001 vs Normal Control; one way ANOVAfollowed by Bonferroni’s posttests

TABLE 5 Effect of treatment on food intake and body weight on Day 4 FoodConsumption (g) Body Weight (g) (day 0 to day 4) (Day 4) % Change MeanSD Mean SD vs Baseline Normal Control 90.5 5.6 491.3 16.9 2.1 Compound 143.25***^(, ##) 2.8 436.2* 21.1 −7.5 Semaglutide 55.73*** 4.1 427.2 42.3−5.3 *p < 0.05, **p < 0.01, ***p < 0.001 vs Normal Control; one wayANOVA followed by Bonferroni’s posttests ^(#)p < 0.05, ^(##)p < 0.01,^(###)p < 0.001 vs Semaglutide; one way ANOVA followed by Bonferroni’sposttests

It was surprisingly found that compounds having X33 as Leu and Ileshowed significant reduction of the blood glucose in the given studieswhereas compounds with amino acids other than Leu and Ile hadsignificantly less effect in lowering the blood glucose. The polypeptideof present disclosure has shown significant reduction in blood glucosewhen compared to the control group. Compounds having amino acids otherthan Leu or Ile at X33 positions were also tested. For example, Leu at32nd position in Compound 1 (SEQ ID NO: 05) was replaced with Lys andSer to obtain compounds Std-1 and Std-2, respectively. Std-1 and Std-2showed only about 35 and 15% reduction in blood glucose AUC_(0-120 min)(Table 6).

TABLE 6 Percentage reduction in Blood Glucose AUC_(0-120 min) in OGTTtest @ 1 mg/Kg dose after 24 hrs. % Glu- % Glu- cose Re- Comp/std coseRe- Comp/std Comp. # duction Semaglutide Comp. # duction LiraglutideStd-1 35.1 0.71 6 75.8 1.83 Std-2 14.8 0.30 7 71.9 1.73 Semaglutide 49.3Liraglutide 41.5

Similarly, the compound 6 which differs from liraglutide in having anadditional Leu at 32nd position and the compound 7 which differs fromliraglutide by having 2nd amino acid Ala replaced with Aib and havingLeu as an additional 32nd amino acid, showed blood glucose loweringeffect at 24 hrs which was significantly higher than liraglutide (Table6).

Once it was determined that the Compound 1 was significantly better interms of glucose reduction, food intake and body weight reduction,experiments were conducted to determine the duration of action of thecompound of the present invention. The effect of the representativecompounds of the present invention (Compound 1, 13 and 16) after 166 hrs(7 days) and 334 hrs (14 days) was studied and compared with that ofsemaglutide. The compounds were tested as per the method provided below:

Animals were divided into three groups—a normal control group, a testgroup and a third semaglutide group, with 4 animals in each group. Theanimals were fasted for 12 hours before initiation of OGTT. To the testgroup animals, Compound 1, Compound 13 and Compound 16 were injectedsubcutaneously at 1 mg/kg dose. To the semaglutide group, a dose of 1mg/kg was injected subcutaneously. After 22 hrs, 166 hrs and 334 hrs ofsubcutaneous injection of test compound or semaglutide, blood glucosewas measured with blood glucose meter (time 0 measurements). All theanimals were then given 2 g/kg of glucose solution orally. Blood glucosewas measured at 20, 40, 60, 90 and 120 minutes following glucosechallenge. Body weight and food intake was recorded. Blood glucose datawas analyzed using Two way ANOVA followed by Bonferroni posttests usingPRISM (Graph Pad version 5.03). Data of Blood glucose AUC_(0-120 min),was analyzed using t test.

Table 7 provides the reduction in AUC of blood glucose for therepresentative compounds of present invention (Compound 1, 13 and 16) incomparison with the control group after 1 day, 7 days and 14 days ofadministration.

TABLE 7 Percentage reduction in Blood Glucose AUC_(0-120 min) in OGTTtest @ 1 mg/Kg dose. Blood Glucose AUC (mg/dL * min) Change in AUCCompound # Time Mean (mg/dL * min) Semaglutide  22 hr 5458.0 −63.4 Exp.1 168 hr 12785.0 −25.5 336 hr 16223.0 −2.4 Comp. 1  22 hr 3173.0 −78.7Exp. 1 168 hr 5941.0 −65.4 336 hr 11920.0 −28.3 Comp. 13  22 hr 2795.0−81.2 Exp. 1 168 hr 6950.0 −59.5 336 hr 11368.0 −31.6 Semaglutide  22 hr4258 −50 Exp. 2 168 hr 7410 −26.6 336 hr 8023.0 −5.5 Comp. 16  22 hr1646 −80.7 Exp. 2 168 hr 4283 −57.6 336 hr 8295.0 −2.3

Compounds 1 and 13 were studied and compared with semaglutide in oneexperiment (Exp. 1) and Compound 16 was studied and compared withsemaglutide in a separate experiment (Exp. 2). After 168 hrs ofinjection, the Compound 1, 13 and 16 of present invention showed about60% reduction in blood glucose AUC when compared to time zero bloodglucose level. On the other hand, semaglutide showed just about 25%reduction in blood glucose level with respect to time zero blood glucoselevel.

Similar observations were made in quantity of food consumed and bodyweight change. As can be seen in the Table 8 below the animalsadministered with the representative compounds (Compound 1, 13 and 16)consumed significantly less food when compared to the animalsadministered with semaglutide. Compound 16 showed substantial loweringof body weight demonstrating potential utility for the treatment ofobesity.

TABLE 8 Effect on food consumption and body weight in OGTT test @ 1mg/Kg dose Body Weight Food Consumption (g) Change (%) Compound # TimeMean Mean Semaglutide  48 h 17.5 −6.5 Exp. 1 154 h 86.3 4.5 324 h 107.911.8 Comp. 1  48 h 8.3 −9.8 Exp. 1 154 h 69.6 5.2 324 h 99.4 9.3 Comp.13  48 h 8.4 −10.2 Exp. 1 154 h 62.3 4.5 324 h 82.44 8.2 Semaglutide  48h 19.3 −8.7 Exp. 2 154 h 50.4 0.2 324 h 96.8 3.8 Comp. 16  48 h 5.9−10.7 Exp. 2 154 h 44.1.0 −7.0 324 h 104.3 −4.3

Example 15: Reduction of HbA1c in db/db Type 2 Diabetic Mice afterChronic Treatment

This study was done in diabetic mouse model. The animals were dividedinto three treatment groups—a diabetic control group, a test group and asemaglutide treatment group. Compound 1 of the present disclosure wasinjected subcutaneously at 0.3 mg/kg dose once a day for 3 days (qd*3)followed by 0.1 mg/kg dose every alternate day for 7 doses (q2d*7)followed by 0.1 mg/kg dose once every four days for two dose cycles(q4d*2). The same dosage regimen was administered in semaglutidetreatment group. Measurements of blood glucose levels and body weightwere done daily. % HbA1c was measured on Day 0, day 7, day 14 and day 27by column chromatography. Cumulative food intake was calculated on day27. % HbA1C data was analyzed by two way ANOVA followed by Bonferroni'sposttests using PRISM (Graph Pad version 5.03).

The test group animals administered with the Compound 1 showedstatistically significant reduction in blood glucose levels as comparedto the diabetic control group (see FIG. 7 ), and the effect was superiorto semaglutide treatment group in latter phase of the study. The testgroup animals administered with Compound 1 showed significant reductionin food intake as can be seen in the results provided in FIG. 8 . FIG. 8provides cumulative food intake from 0 to 27^(th) day by control anddb/db mice treated with the test compound. Both the test compound andsemaglutide showed a statistically significant reduction in food intakeas compared to the diabetic control group. Further, the test compoundshowed a significantly lower food intake, as compared to semaglutide. Inthe same study, the Compound 1 has also shown significant reduction inbody weight when compared to diabetic control group. FIG. 9 provides theresult of % change in body weight for control and test group from day 0to day 27. The test Compound 1 showed a significant reduction of −16% ascompared to −8% as observed in semaglutide treatment group (see FIG. 09).

In diabetes mellitus, higher amounts of HbA1c, indicating poorer controlof blood glucose levels, have been associated with cardiovasculardisease, nephropathy, neuropathy, and retinopathy. In a 27 day study,Compound 1 showed statistically significant reduction in HbA1c level indb/db type 2 diabetic mice after chronic treatment. Table 9 below andFIG. 10 provide the level of HbA1c at 0 and 27 days in Diabetic controlgroup and group after chronic treatment with the Compound 1. The effectwas statistically significant even when compared to semaglutide.

TABLE 9 Effect of treatment on % HbA1c levels in db/db mice % HbA1c TimeCompound 1 Semaglutide Point Diabetic Control (DC) % Change % Change(Days) Mean SD n Mean SD n vs DC Mean SD n vs DC 0 4.70 0.52 6 4.87 0.596 — 5.05 0.35 5 — 27 7.30 1.17 6 3.73***^(##) 0.86 6 −3.57 5.03*** 0.695 −2.26 *p < 0.05, **p < 0.01, ***p < 0.001 vs Diabetic Control; two wayANOVA followed by Bonferroni's posttests ^(#)p < 0.05, ^(##)p < 0.01,^(###)p < 0.001 vs Semaglutide; one way ANOVA followed by Bonferroni'sposttests

In a separate study, test Compound 1, 13 and 16 were studied andcompared with semaglutide for their effect on HbA1c & insulin level andcumulative food consumption, body weight change and blood glucose AUC.The study was performed in similar manner as above on diabetic mousemodel. The animals were divided into three treatment groups—a diabeticcontrol group, a test group and a semaglutide treatment group. Therepresentative compounds of the present disclosure, Compound 1, Compound13 and Compound 16, were injected subcutaneously at 3.04 or 6.078 nMdose (every alternate day up to day 28 (q2d*15). The same dosage regimenwas administered in semaglutide treatment group. Measurements of bloodglucose levels and body weight were done daily. % HbA1c, Insulin wasmeasured on Day 0, day 14 and day 29. Cumulative food intake and bodyweight change was calculated on day 14 and 29. % HbA1c, Insulin data wasanalyzed by two way ANOVA followed by Bonferroni's posttests using PRISM(Graph Pad version 5.03). Whereas Blood Glucose AUC, Body weight changeand Cumulative food intake data was analyzed by one way ANOVA followedby Bonferroni's posttests using PRISM (Graph Pad version 5.03). From day29 to day 45, animals were kept on recovery period during which no drugtreatment was given. Blood Glucose and Body weight was measured duringthis period. On day 45, Body weight Changes, % HbA1c, Insulin wasmeasured.

The results are provided in Tables 10, 11 and 12 below.

TABLE 10 HbA1C (%): Compound 1 (6.078 nM), Compound 13 (3.04 & 6.078nM), Compound 16 (3.04 & 6.078 nM); (q2d * 15) (n = 7) % HbA1C DiabeticCompound 1, Compound 13, Compound 13, Compound 16, Compound 16,Semaglutide, Time Control 6.078 nM, 3.04 nM, 6.078 nM, 3.04 nM, 6.078nM, 12.15 nM, Point (DC) q2d * 15 q2d * 15 q2d * 15 q2d * 15 q2d * 15q2d * 15 (Days) Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD0 7.7 1.2 7.8   0.8 7.7   1.5 7.83 0.76 7.74 1.27 7.83 0.99 7.66 1.13 148.0 1.0 6.4**  0.5 6.6*  0.9 6.00*** 0.53 5.67***# 0.68 5.64***# 0.497.04 1.15 29 8.3 1.0 6.2*** 0.5 6.4*** 0.8 5.64*** 0.51 5.33***# 0.395.16***# 0.24 6.47*** 1.03 *= p < 0.05, **= p < 0.01, ***= p < 0.001 vs.Diabetic Control, #p < 0.05, ##p < 0.01, ###p < 0.001 vs Semaglutide;One way ANOVA followed by Bonferroni's posttests

TABLE 11 Insulin (ng/mL): Compound 1 (6.078 nM), Compound 13 (3.04 &6.078 nM), Compound 16 (3.04 & 6.078 nM); (q2d * 15) (n = 7) Insulin(ng/mL) Compound 1, Compound 13, Compound 13, Compound 16, Compound 16,Sema, Time Diabetic 6.078 nM, 3.04 nM, 6.078 nM, 3.04 nM, 6.078 nM,12.15 nM, Point Control q2d * 15 q2d * 15 q2d * 15 q2d * 15 q2d * 15q2d * 15 (Days) Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD0 17.6 5.5 18.0 5.8 21.7 12.1 26.2 14.0 13.9 6.6 21.8 9.6 18.7 10.3 1423.0 10.7 58.1** 31.6 41.8 28.2 61.2*** 23.7 50.8* 27.7 58.1** 12.5 37.526.5 29 16.9 6.6 54.7** 6.4 54.4** 27.0 59.4*** 22.4 53.8** 23.5 68.2***27.1 48.1** 25.3 *= p < 0.05, **= p < 0.01, ***= p < 0.001 vs DiabeticControl, #p < 0.05, ##p < 0.01, ###p < 0.001 vs Semaglutide; One wayANOVA followed by Bonferroni posttests

TABLE 12 Cumulative food Consumption, Blood Glucose AUC_((mg/dL*days))and Body Weight Changes: Compound 1 (6.078 nM), Compound 13 (3.04 &6.078 nM), Compound 16 (3.04 & 6.078 nM); (q2d * 15) Cumulative FoodBlood Glucose AUC Body Weight Change Intake (g) (Day 0-29) (mg/dL *days) Day 29, Groups Day 0-29 % Change Vs % Change vs Day 0 (n = 7) MeanSD Mean SD Control Mean SD Diabetic 99.3 37.16 16948.1 281.48 9.4 6.4Control Compound 1, 79.8 25.09 9194.6*** 450.84 −45.75 −0.3 2.2 6.078 nMCompound 13, 75.2 18.45 9515.4*** 805.21 −43.86 1.2 3.8 3.04 nM Compound13, 70.4 10.85 8451.7***## 612.94 −50.13 −1.1 2.4 6.078 nM Compound 16,77.4 23.82 9399.0*** 680.38 −44.54 −1.4 5.4 3.04 nM Compound 16, 66.729.48 8086.1***## 623.80 −52.29 −5.5 3.1 6.078 nM Semaglutide, 92.0 6.649867.9*** 832.73 −41.78 0.7 3.9 12.155 nM *= p < 0.05, **= p < 0.01,***= p < 0.001 vs Diabetic Control, #p < 0.05, ##p < 0.01, ###p < 0.001vs Semaglutide; One way ANOVA followed by Bonferroni posttests

The representative compounds of the present disclosure (Compound 1, 13and 16) at about 3 nM and 6 nM dose showed significant reduction inHbA1c, blood glucose, food consumption and body weight when compared tocontrol (Table 12). The reduction was comparable to that shown bysemaglutide at about 12 nM dose. Moreover, the effect was seen evenafter 29 days (Tables 13 and 14) which demonstrates the potential ofcompounds of present invention for developing long acting drug which donot require frequent administration and hence adding to the patientcompliance.

TABLE 13 Recovery study-Blood Glucose AUC_((mg/dL*days)) Blood GlucoseAUC Blood Glucose AUC Groups (Day 30-37) (mg/dL * days) % Change (Day38-45) (mg/dL * days) % Change vs (n = 3) Mean SD vs Control Mean SDControl Diabetic 3591.7 47.04 3601.7 38.66 Control Compound 1, 2787.3*17.67 −22.39 3335.7 66.11 −7.39 6.078 nM Compound 13, 2588.7** 411.97−27.93 3401.0 61.00 −5.57 3.04 nM Compound 13, 2875.7 436.87 −19.943361.0 205.83 −6.68 6.078 nM Compound 16, 2420.0** 89.71 −32.62 3284.0*81.66 −8.82 3.04 nM Compound 16, 2167.7*** 210.19 −39.65 3134.7** 103.39−12.97 6.078 nM Semaglutide, 3032.0 182.40 −15.58 3415.7 88.82 −5.1612.155 nM *p < 0.05, **p < 0.01, ***p < 0.001 vs Diabetic Control and #p< 0.05, ##p < 0.01, ###p < 0.001 vs Semaglutide; One Way ANOVA followedby Bonferroni's posttests

TABLE 14 Recovery study-% HbA1C and Insulin (ng/mL) Delta Insulin BodyWeight % Groups % HbA1C HbA1C (ng/mL) Change vs Day 45 (n = 3) Mean SDvs. DC Mean SD Mean SD Diabetic 8.5 0.4 20.6 12.0 7.8 1.08 ControlCompound 1, 7.6 0.5 −0.9 30.9 8.9 5.9 3.68 6.078 nM Compound 13, 7.5 0.4−1.0 31.2 32.7 6.6 0.86 3.04 nM Compound 13, 7.3 0.4 −1.2 34.3 12.4 5.52.19 6.0788 nM Compound 16, 7.1* 0.2 −1.4 15.5 8.2 6.1 0.38 3.04 nMCompound 16, 7.2* 0.5 −1.3 18.4 18.7 5.7 0.36 6.078 nM Semaglutide, 7.80.3 −0.7 19.7 4.2 9.5 4.68 12.155 nM *p < 0.05, **p < 0.01, ***p < 0.001vs Diabetic Control and #p < 0.05, ##p < 0.01, ###p < 0.001 vsSemaglutide; One Way ANOVA followed by Bonferroni's posttests

These results demonstrate that the compound of present invention canfind potential use for the treatment of diabetes and obesity.

We claim:
 1. A Glucagon-like Peptide-1 (GLP-1) agonist peptide or saltthereof comprising a modification, wherein the modification is a groupof formula (I) attached to an amino acid residue in the peptide

wherein U represents —C(O)—CH₂—O—(CH₂)₂—O—(CH₂)₂—NH—} wherein } is pointof attachment to W; W represents —C(O)—NH—(CH₂)₃₋₄—NH—],—C(O)—C(CH₃)₂—NH—], or

wherein ] is point of attachment to Y; Y represents—C(O)—(CH₂)₂—CH(COOH)NH— and — is point of attachment to Z; Z represents—C(O)—(CH₂)_(n)—COOH or —C(O)—(CH₂)_(n)—CH₃ wherein n is an integer from14-20.
 2. The peptide according to claim 1, wherein the group of formula(I) is attached to a lysine residue in the peptide.
 3. The peptideaccording to claim 1, wherein W represents —C(O)—NH—(CH₂)₃₋₄—NH—] or—C(O)—C(CH₃)₂—NH—].
 4. The peptide according to claim 1, wherein U-W-Y-Zrepresents a group of the formula:

wherein n is an integer from 14-20 and Ro is CO₂H or CH₃.
 5. The peptideaccording to claim 4, wherein n is an integer from 16 to
 18. 6. Thepeptide according to claim 4, wherein n is 16 to 18 and R_(o) is CO₂H.7. A pharmaceutical composition comprising a Glucagon-like Peptide-1(GLP-1) agonist peptide or salt thereof according to claim 1, togetherwith one or more pharmaceutically acceptable excipients.